The present invention relates to a novel, human adipose tissue-derived polypeptide having water channel activity and to a DNA sequence encoding for the polypeptide.
The permeation of water through a cell membrane generally occurs slowly by way of diffusion into the lipid bilayer which is the main structure of the cell membrane. Recently, however, it was discovered that, in certain kinds of cells, water is transferred rapidly through the cell membrane, suggesting the involvement in the above phenomenon of some membrane protein selectively permeable to water. Thereafter, such membrane proteins of various kinds have actually been isolated. Such membrane proteins are designated as water channels. In this specification, the function of the above water channels which has selective permeation of water through the cell membrane is referred to as xe2x80x9cwater channel activityxe2x80x9d. The water channels may be permeable to water alone or permeable to not only water but also low-molecular-weight substances such as glycerol and urea.
As a membrane protein having such water channel activity, there have been isolated a group of membrane proteins known as aquapolins (AQPs). Furthermore, some aquapolin genes have so far been cloned, and aquapolins such as AQP1 through AQP5, FA-CHIP and AQP-xcex3TIP have been discovered in mammals, amphibians, plants, etc. [cf. e.g. Akira Sasaki, Igaku no Ayumi (Advances in Medicine), vol. 173, No. 9, 1995].
P. Agre et al. reported, in Science (vol. 256, pp. 385 to 387, 1992) that Xenopus laevis oocytes in which the in vitro transcript RNA for CHIP28, the current designation of which is AQP1, had been introduced showed increased water permeability. In Science (vol. 264, pp. 92 to 95, 1994), B. A. van Oost et al. disclosed the amino acid sequence of human AQP2 and suggested that this should be involved in vasopressin-dependent urine concentration.
In Proc. Natl. Acad. Sci. USA (vol. 91, pp. 6269 to 6273, 1994), Ishibashi et al. disclosed the nucleotide sequence of the gene for renal collecting tubule-derived AQP3 and the amino acid sequence encoded thereof. Ishibashi et al. confirmed its water channel activity by injecting the AQP3 cRNA into Xenopus laevis oocytes and measuring the water permeability thereof. Ishibashi et al. reported that this AQP3 transported not only water but also nonionic small molecules such as urea and glycerol.
In Proc. Natl. Acad. Sci. USA (vol. 91, pp. 13052 to 13056, 1994), J. S. Jung et al. reported about the isolation of AQP4. This AQP4 is known to occur most abundantly in mammalian brains and have mercury resistance. In J. Biol. Chem. (vol. 270, pp. 1908 to 1912, 1995), S. Raina et al. who prepared rat salivary gland-derived AQP5 cDNA describe the nucleotide sequence of the cDNA and the amino acid sequence encoded thereby. S. Raina et al. cloned the cDNA by utilizing the occurrence of an NPA sequence and confirmed its function by observing that the cRNA enhances the water permeability of Xenopus laevis oocytes.
The aquapolin family mentioned above is considered to be involved in water metabolism in mammals and, for example, it has been confirmed that AQP2 is found only in the renal collecting tubule luminal membrane, which is indicative of its close association with the vasopressin-urea concentration system, and its involvement in renal diseases has become acknowledged. Therefore, such membrane proteins having water channel activity are of importance in any attempt to develop novel therapies for water-associated diseases.
Meanwhile, the expression of the aquapolin family mentioned above has been confirmed in such organs as kidney, brain, gall bladder, eye, intestine, salivary gland and bronchus but there is no report as yet about the occurrence of membrane proteins having water channel activity in other organs or tissues, particularly in adipose tissue.
In view of the above-mentioned state of the art, the present invention has for its object to provide a novel membrane protein having water channel activity and a DNA sequence encoding for the polypeptide.
The present invention is related to a novel polypeptide having water channel activity which has the amino acid sequence, within the molecule thereof, shown in the sequence listing under SEQ ID NO:1.
The present invention is also related to a nucleotide sequence itself which codes for a polypeptide having, within the molecule thereof, the amino acid sequence shown in the sequence listing under SEQ ID NO:1 and having water channel activity.
The present invention is further related to the DNA sequence shown in the sequence listing under SEQ ID NO:2.
The present invention is still further related to a polypeptide having water channel activity which has the amino acid sequence, within the molecule thereof, encoded by the nucleotide No. 173 to No. 1198 of the nucleotide sequence shown in the sequence listing under SEQ ID NO:2.
In the following, the present invention is described in detail.
The polypeptide of the present invention has the amino acid sequence shown in the sequence listing under SEQ ID NO:1. This polypeptide has a sequence composed of three amino acids, namely asparagine-proline-alanine, as the amino acid Nos. 195 to 197. However, the characteristic feature common to the so-far known AQPs, that said asparagine-proline-alanine sequence occurs twice, is not found in the polypeptide of the present invention. That this polypeptide has water channel activity can be confirmed from the fact that it enhances the water permeability of Xenopus laevis oocytes.
The above polypeptide may be generated by translation by a protein synthesis system constituted, in vivo or in vitro, based on the nucleotide sequence coding for the amino acid sequence of said polypeptide. The nucleotide sequence of the present invention substantially has a region coding for the amino acid sequence of said polypeptide and, where necessary, may contain one or more other regions such as a promoter region. In the protein synthesis based on genetic information, the information carried by the gene DNA is transcribed into mRNA as the result of DNA-dependent RNA synthesis aided by RNA polymerase. And, this mRNA is translated into the amino acid sequence in a tRNA-containing protein synthesis system. Therefore, the nucleotide sequence of the present invention includes not only the DNA sequence but also the RNA sequence. Furthermore, since it is generally known that, for an amino acid, there is one or a plurality of codons corresponding thereto, it is a matter of course that the above-mentioned nucleotide sequence is not limited to only one sequence but may include nucleotide sequences resulting from substitution of another synomyous codon coding for the same amino acid.
The above polypeptide can be formed based on the genetic information carried by the DNA sequence shown in the sequence listing under SEQ ID NO:2. This polypeptide is encoded by that portion of the nucleotide sequence shown in the sequence listing under SEQ ID NO:2 which ranges from the nucleotide No. 173 to No. 1198. Of the DNA sequence shown in the sequence listing under SEQ ID NO:2, the nucleotide sequences other than the portion of said nucleotide numbers are noncoding regions, among which the polyadenylation consensus sequence AATAAA occurs at the nucleotide No. 1234 to No. 1239. Other possible reading frames of said DNA sequence shown under SEQ ID NO:2 can be excluded from consideration, since the polypeptides encoded are very small-sized, hence considered to be incapable of performing any water channel function.
It has been confirmed by the inventors that the full-length sequence of the above nucleic acid bases has no counterpart sequence either in GenBank or in dbEST.
The polypeptide of the present invention has water channel activity in adipose tissue. While adipose tissue is distributed in various parts of the living organism, the polypeptide of the present invention has an action to control the transfer of water in such adipose tissue and is expected to be effective in upholding normal functions of adipose tissue at various sites.
The above-mentioned DNA sequence given under SEQ ID NO:2 corresponds to the nucleotide sequence of cDNA obtained from human adipose tissue by cloning. Human adipose tissue is a tissue which stores fat as energy reserves. It is known that various proteins are formed in this adipose tissue. A 3xe2x80x2-directed DNA library is known as a cDNA library from which the genes actually expressed in this adipose tissue or, in other words, the mRNA composition in this adipose tissue can be copied faithfully. This 3xe2x80x2-directed DNA library contains only those specified 3xe2x80x2-terminal regions of mRNAs which range from poly(A) to the MboI site which is a restriction enzyme recognition site upstream of said poly(A) and, therefore, said library is suited for template preparation by the PCR technique. Therefore, by extracting a clone from this library and using it to determine a longer nucleotide sequence including the amino acid coding region from this complete adipose tissue cDNA library, it becomes possible to obtain the genetic information concerning the protein which is actually formed in adipose tissue. The DNA sequence of the present invention as shown under the above-mentioned SEQ. ID. NO:2 is found by such cloning. A method of obtaining the cDNA by cloning from human adipose tissue is now describe in detailed.
Known as said method is, for example, the method described in Biochem. Biophys. Res. Commun., 221, 286 to 289 (1996). According to this method, the total RNA is first separated from adipose tissue and, when necessary, purified to give poly(A) RNA. For this purification, commercially available purification kits can be used. For example, Pharmacia""s Quick prep mRNA purification kit or the like in which oligo(dT)-cellulose and various buffers are used in combination can judiciously be employed. Then, a double-stranded cDNA is synthesized using a pUC19 system vector primer and the double-stranded cDNA so synthesized is selectively cleaved with the restriction enzyme MboI (which recognizes the nucleotide sequence GATC). On that occasion, the GATC sequence on the vector molecule side, which can be methylated to give GmATC when replication is effected in dam+ bacterial cells, is not cleaved with MboI. As the cleaved cDNA is subjected to self-cyclization using E. coli ligase, a plasmid containing a cDNA fragment extending from poly(A) to the nearest MboI site is completed. This plasmid is introduced into Escherichia coli, followed by cultivation and selection of a transformant E. coli colony. Then, the cDNA in said colony is amplified by the PCR technique using appropriate PCR primers.
On the other hand, the full-length double-stranded cDNA synthesized using the pUC19 system vector primer is cleaved at the 5xe2x80x2 end using T4 polymerase and subjected to cyclization using T4 ligase and introduction into Escherichia coli for transformation. From among the thus-obtained transformant colonies, the desired colony is obtained by screening using, as a probe, a labeled form of the adipose tissue-specific cDNA obtained from said 3xe2x80x2-directed DNA library by the method mentioned above. The insert cDNA in this colony is amplified by the PCR technique using appropriate PCR primers. The amplification product is purified and, after sonication, subcloned into the M13 phage.
The nucleotide sequence of the thus-cloned cDNA can be determined, for example, by reaction with a primer dye, purification and analysis using an automated sequencer or the like. In this manner, the DNA sequence of the present invention can be obtained.
The polypeptide of the present invention has water channel activity. This water channel activity can be confirmed by observing an enhancement of water permeability in Xenopus laevis oocytes. It is known that no AQP family gene has been expressed in Xenopus laevis oocytes and, therefore, any increase in water permeability as caused by the injected mRNA can be easily confirmed. For this reason, said oocytes are widely used in confirming water channel activity. For example, in Proc. Natl. Acad. Sci. USA, vol. 91, pp. 6269 to 6273 (1994), Ishibashi et al. confirmed the water channel activity by inserting the AQP3 cDNA into the pSP64T-derived BlueScript vector, synthesizing the cRNA using T7 RNA polymerase, injecting this cRNA into Xenopus laevis oocytes and, after 48 to 62 hours of incubation following injection, observing an increase in water permeability and in the volume of the oocyte.
The polypeptide encoded by the DNA sequence of the present invention can be identified by analyzing the amino acid sequence of the polypeptide synthesized in an Escherichia coli protein synthesis system constituted in vitro. In this case, the methods of identifying N- and C-terminal sequences of expression products as described in Shin Seikagaku Jikken Koza (Experiments in Biochemistry, A New Course) 1 (published by Tokyo Kagaku Dojin), pages 22 to 24 can be employed.
The following examples illustrate the present invention in further detail. These examples are, however, by no means limitative of the scope of the present invention.